Hydroxytyrosol is a plant secondary metabolite which can be found almost exclusively in leaves and fruit of olive (Olea europaea L.). It is especially abundant in olive mill wastewaters [Fernández-Bolaños, Olive Oil—Constituents, quality, health properties and bioconversions. Ed. Boskow Dimitrios, ISBN: 978-953-307-921-9. Chapter 20]. Hydroxytyrosol has been reported to be present in grapes (Vitis vinifera L.), although in a smaller proportion [Fernández, M I., et al., “Bioactive compounds in wine: Resveratrol, hydroxytyrosol and melatonin: A review”, Food Chemistry, 130 (2012), pp. 797-813].
Hydroxytyrosol is a phenyl ethyl alcohol, 2-(3,4-dihydroxyphenyl)ethanol. It is a metabolite of oleuropein (ester of hydroxytyrosol, elenolic acid and glucose), from which it can be obtained by hydrolysis [Omar, S H., “Cardioprotective and neuroprotective roles of oleuropein in olive”, Saudi Pharmaceutical Journal, 18 (2010), pp. 111-121].
Hydroxytyrosol's derivatives include tyrosol, oleuropein, verbascoside and related compounds derived from simple phenolic acids, such us gallic acid and others. Notable examples of verbascoside related compounds are those belonging to the phenylpropanoid glycoside group which are structurally characterized by the presence of caffeic acid moieties and 2-(3,4-dihydroxyphenyl)ethanol (hydroxytyrosol) moieties bound to a β-(D)-glucopyranoside [Vertuani, S., “Activity and Stability Studies of Verbascoside, A Novel Antoxidant, in Dermo-Cosmetic and Pharmaceutical Topical Formulations”, Molecules, 16 (2011), pp. 7068-7080]. Due to its chemical structure, verbascoside (also known as acteoside) is highly soluble in hydrophilic solutions, such as methanol or hydroalcoholic mixtures. Verbascoside related compounds include echinacoside, martynoside, leucosceptoside, and other related phenylpropanoids.
Verbascoside is a secondary metabolite in plants and verbascoside related compounds are widely distributed in nature. Main sources include black horehound (Ballota nigra L.), pale purple cone-flower (Echinacea pallida (NUTT.)), devil's claw (Harpagophytum procumbens (BURCH)), bitter melon (Momordica charantia L.), olive (Olea europaea subsp. Europaea), figworts (Scrophularia scorodonia L.), Baikal skullcap (Scutellaria baicalensis (GEORGI)), sesame (Sesamum indicum L.) and common mullein (Verbascum thapsus L.) [Source Dr. Duke's Phytochemical and Etnobotanical Database]. Thus, verbascoside related compounds may be either chemically synthesized or obtained from the corresponding plant extracts.
Pentacyclic triterpenes are secondary plant metabolites widespread in plants, mainly in peel, leaves and stem bark. They are part of the chemical family of triterpenoids, polycyclic structures of thirty atoms of carbon. Triterpenoids are mainly subdivided in three families depending on their skeletal structures: lupane, oleane and ursane (see Table 1 below). In virtue of their structure, pentacyclic triterpenoids present very low solubility in hydrophilic solvents such as water. In the other hand, their solubility in organic solvents such us acetone or methanol has been demonstrated to be moderate-to-high [Liu, L., et al., “Solubility of Oleanolic Acid in Various Solvents from (288.3 to 328.3) K”, Journal of Chemical and Engineering Data, 52 (2007), pp. 2527-2528].
Pentacyclic triterpenes are widely distributed in nature. Oleanolic acid is present in olive leaves (Olea europaea L.), marigold flowers (Calendula officinalis L.), rosemary leaves (Rosmarinus officinalis L.) and clove flowers (Syzygium aromaticum L.); maslinic acid is present in bearberry leaves (Arctostaphylos uva-ursi L.) and olive fruit (O/ea europaea L.); betulin is present in birch bark (Betula alba L.); betulinic acid is present in eucalyptus leaves (Eucalyptus), planes bark (Platanus acerifolia), rosemary leaves (Rosmarinus officinalis L.) [Jäger, S. et al., “Pentacyclic Triterpene Distribution in Various Plants—Rich sources for a New Group of Multi-Potent Plant Extracts”, Molecules, 14 (2009), pp. 2016-2031] and clove leaves (Syzygium aromaticum L.) [Aisha, A F A., et al., “Syzigium aromaticum extracts as good source of betulinic acid and potential anti breast cancer”, Brazilian Journal of Pharmacognosy, 22 (2012), pp. 335-343].
Hydroxytyrosol and related polyphenols are known as potent anti-oxidants and cytoprotectors. Different scientific articles and patents disclose their neuroprotective properties and its main target is believed to be related to mitochondrial targeting, one of the cell components mainly involved in neurodegenerative diseases [Schaffer S. “Hydroxytyrosol-rich olive wastewater extract protects brain cells in vitro and ex vivo” J. Agricul. Food Chem., 55 (2007), pp. 5043-5049]. Among other studies, hydroxytyrosol and related phenols have been shown to protect neurons against hypoxia-reoxygenation [González-Correa JA. “Neuroprotective effect of hydroxytyrosol and hydroxytyrosol acetate in rat brain slices subjected to hypoxia-reoxygenation” Neurosci Lett., 446 (2008), pp. 143-6.], act as Tau protein aggregation inhibitors in Alzheimer's disease [Daccache A. “Oleuropein and derivatives from olives as Tau aggregation inhibitors” Neurochem Int., 58 (2011), pp. 700-7] or reducing oxidative damage in an animal model of Huntington disease [Tasset I. “Olive oil reduces oxidative damage in a 3-nitropropionic acid-induced Huntington's disease-like rat model” Nutr Neurosci., 14 (2011), pp. 106-11]. Their use for neuroprotection has also been extensively described in different patents applications [US 2003/0236202A1, WO 2006/114467 A1, and US 2010/0130621 A1]. These derivatives have been also shown to exhibit anti-inflammatory effects that can mediate indirect neuroprotection [Zhang X. “Hydroxytyrosol inhibits pro-inflammatory cytokines, iNOS, and COX-2 expression in human monocytic cells” Naunyn Schmiedebergs Arch Pharmacol., 379 (2009), pp. 581-6].
Verbascoside, which is mainly extracted from plants, also displays a neuroprotective profile, acting as anti-oxidant, cytoprotector and anti-inflammatory agent [Wang H Q. “Upregulation of heme oxygenase-1 by acteoside through ERK and PI3 K/Akt pathway confer neuroprotection against beta-amyloid-induced neurotoxicity” Neurotox Res., 21 (2012), pp. 368-78; Koo K A. “Acteoside and its aglycones protect primary cultures of rat cortical cells from glutamate-induced excitotoxicity” Life Sci., 79 (2006), pp. 709-16; Esposito E. “Protective effect of verbascoside in activated C6 glioma cells: possible molecular mechanisms” Naunyn Schmiedebergs Arch Pharmacol., 381 (2010), pp. 93-105].
Triterpenic acids derived from plant extracts such as oleanolic acid or maslinic acid have been also extensively known for their neuroprotective and anti-neuroinflammatory properties [Martín E. “Beneficial actions of oleanolic acid in an experimental model of multiple sclerosis: A potential therapeutic role”, Biochem Pharmacol, 79 (2010), pp. 198-208], primarily targeting neuronal apoptotic pathways and inhibiting neuroinflammatory signals such as microglial nitric oxide synthesis [Qian Y. “Maslinic acid, a natural triterpenoid compound from Olea europaea, protects cortical neurons against oxygen-glucose deprivation-induced injury” Eur J Pharmacol., 670 (2011), pp. 148-53.; Cho S O. “Aralia cordata protects against amyloid beta protein (25-35)-induced neurotoxicity in cultured neurons and has antidementia activities in mice” J Pharmacol Sci., 111 (2009), pp. 22-32]. These properties of triterpenoids or derivatives thereof have also been described in patent applications such as WO 2011/015692 A2.
Products comprising a combination of pentacyclic triterpenes and hydroxytyrosol or derivatives thereof have been disclosed, for example in WO 2007/096446 A1 and EP2 033 620 A1. Said applications disclose the use of said product combinations as a pronutrient in animal feed and for the preparation of a cream, respectively. However, these documents fail to disclose or suggest the use of such product combination for the protection of neurons and also fail to disclose a product comprising 3,4-dihydroxyphenylglycol.
As explained above, pentacyclic triterpenes, hydroxytyrosol or derivatives thereof have shown neuroprotective effectiveness when used individually. Surprisingly, the inventors have now discovered that the combination of specific pentacyclic triterpenes and hydroxytyrosol or specific derivatives thereof, such as verbascoside, can protect neurons in a more potent way than just the sum of the individual effects, i.e. the combination of specific pentacyclic triterpenes of formula (I) and hydroxytyrosol or specific derivatives thereof (having formula (II)), such as verbascoside, has a synergistic effect in the protection of neurons in particular when the weight of the compounds of formula (I) with respect to the weight of the compounds of formula (II) is in the range from 20:1 to 1:10.